Muzaffar A. Anwar

Untargeted UPLC-MS Profiling Pipeline to Expand Tissue Metabolome Coverage: Application to Cardiovascular Disease

Metabolic profiling studies aim to achieve broad metabolome coverage in specific biological samples. However, wide metabolome coverage has proven difficult to achieve, mostly because of the diverse physicochemical properties of small molecules, obligating analysts to seek multiplatform and multimethod approaches. Challenges are even greater when it comes to applications to tissue samples, where tissue lysis and metabolite extraction can induce significant systematic variation in composition. We have developed a pipeline for obtaining the aqueous and organic compounds from diseased arterial tissue using two consecutive extractions, followed by a different untargeted UPLC-MS analysis method for each extract. Methods were rationally chosen and optimized to address the different physicochemical properties of each extract: hydrophilic interaction liquid chromatography (HILIC) for the aqueous extract and reversed-phase chromatography for the organic. This pipeline can be generic for tissue analysis as demonstrated by applications to different tissue types. The experimental setup and fast turnaround time of the two methods contributed toward obtaining highly reproducible features with exceptional chromatographic performance (CV % < 0.5%), making this pipeline suitable for metabolic profiling applications. We structurally assigned 226 metabolites from a range of chemical classes (e.g., carnitines, α-amino acids, purines, pyrimidines, phospholipids, sphingolipids, free fatty acids, and glycerolipids) which were mapped to their corresponding pathways, biological functions and known disease mechanisms. The combination of the two untargeted UPLC-MS methods showed high metabolite complementarity. We demonstrate the application of this pipeline to cardiovascular disease, where we show that the analyzed diseased groups (n = 120) of arterial tissue could be distinguished based on their metabolic profiles.

A Review of Familial, Genetic, and Congenital Aspects of Primary Varicose Vein Disease

Varicose veins are a common manifestation of chronic venous disease (CVD) and present as excessively dilated, tortuous, and elongated superficial veins in the lower limbs. Varicose veins arise either secondary to vein wall remodeling or valvular incompetence leading to blood stasis and venous hypertension.1 Patients may experience lower limb pain, muscle cramps, bleeding, swelling, and skin changes, which include lipodermatosclerosis and eventual ulceration.2 To date, no specific cause for the development of varicose veins has been identified. However, various genetic and environmental risk factors have been ascribed to their formation. Primary varicose veins have been shown to affect up to one third of the Western adult population and are a major cause of morbidity. Estimates of prevalence range from 2% to 56% in men and 1% to 60% in women.3,4 This inconsistency between reports may be due to heterogeneity of study populations, study designs, and experimental methodologies used as well as actual variations in the general population. Some reports suggest that varicose veins are more common in women than men,5–8 whereas others have shown the opposite.2,3,9,10 A cross-sectional survey in Edinburgh, UK, involving a total of 1566 patients, showed an age-adjusted prevalence of Grade 1 truncal varices in 33.3% of men compared with 26.2% of women.10 It has also been reported that there is a significant correlation between CVD onset and sex with females showing first symptoms at a mean age of 30.8 years and males at 36.8 years.8 Besides sex, the prevalence of varicose veins increases with age. The Edinburgh Vein Study reported the prevalence of varicose veins increases from 11.5% in 18 to 24 year olds age to 55.7% in 55 to 64 year olds.10 Similarly, other studies have reported …

In-vitro Identification of Distinctive Metabolic Signatures of Intact Varicose Vein Tissue via Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

OBJECTIVES Nuclear magnetic resonance (NMR) spectroscopy is an established tool for metabolic profiling of tissues or biofluids with utility in identifying disease biomarkers and changes in enzymatic or gene expression. This pilot study aims to compare the metabolic profiles of intact varicose and non-varicose vein tissue via magic angle spinning (MAS) NMR spectroscopy with a view to promoting the understanding of the pathogenesis of varicose vein formation. METHODS Varicose vein tissue (n = 8) was collected from patients undergoing varicose veins surgery. Control non-varicose great saphenous vein samples were collected from patients undergoing lower limb amputation (n = 3) and peripheral arterial bypass surgery (n = 5). Intact tissue samples (average weight 10.33 ± 0.8 mg) from each vein segment were analysed using 1D MAS (1)H NMR (600 MHz) spectroscopy. For selected vein samples, two-dimensional (2D) NMR experiments were performed. Differences between spectra from varicose and non-varicose tissues were elucidated using a variety of multivariate statistical analyses. RESULTS The metabolic profiles of varicose veins samples were clearly differentiated from non-varicose veins samples. Lipid metabolites were present at a higher concentration in the non-varicose veins group whilst creatine, lactate and myo-inositol metabolites were more characteristic of the varicose veins group. CONCLUSION We demonstrate differential metabolic profiles between varicose veins and non-varicose veins. Elucidating the metabolic signature underlying varicose veins can further improve our understanding of the biological mechanisms of disease initiation, progression, and aid in identifying putative therapeutic targets.

Comparison of disease-specific quality of life tools in patients with chronic venous disease.

Objectives This work was presented as a poster in the American Venous Forum 25th Annual Meeting; 28 February 2013; Phoenix, Arizona, USA. Quality of life (QoL) is an important outcome measure in the treatment for chronic venous disease. The Aberdeen Varicose Vein Questionnaire (AVVQ) and the ChronIc Venous Insufficiency quality of life Questionnaire (CIVIQ-14) are two validated disease-specific QoL questionnaires in current use. The aim of this study is to evaluate the relationship between the AVVQ and the CIVIQ-14 to enable better comparison between studies and to compare these disease-specific QoL tools with generic QoL and clinician-driven tools. Methods Adults attending our institution for management of their varicose veins completed the AVVQ, CIVIQ-14 and EuroQol-5D (EQ-5D). Clinical data, CEAP classification and the Venous Clinical Severity Score (VCSS) were collected. The relationship between the AVVQ and CIVIQ-14 scores was analysed using Spearman’s correlation. The AVVQ and CIVIQ-14 scores were also analysed with a generic QoL tool (EQ-5D) and a clinician-driven tool, the VCSS. Results One hundred patients, mean age 57.5 (44 males; 56 females), participated in the study. The median AVVQ score was 21.9 (range 0–74) and the median CIVIQ-14 score was 30 (range 0–89). A strong correlation was demonstrated between the AVVQ and CIVIQ-14 scores (r = 0.8; p < 0.0001). Strong correlation was maintained for patients with C1-3 disease (r = 0.7; p < 0.0001) and C4-6 disease (r = 0.8; p < 0.0001). The VCSS correlated strongly with the AVVQ and CIVIQ-14 scores (r = 0.7; p < 0.0001 and r = 0.7; p < 0.0001, respectively). Both the AVVQ and CIVIQ-14 scores correlated well with the EQ-5D score (r = −0.5; p < 0.0001 and r = −0.7; p < 0.0001, respectively). Conclusions This study demonstrates that there is good correlation between two widely used varicose vein specific QoL tools (AVVQ and CIVIQ-14) across the whole spectrum of disease severity. Strong correlation exists between these disease-specific QoL tools and generic and clinician-driven tools. Our findings confirm valid comparisons between studies using either disease-specific QoL tool.

Optimization of metabolite extraction of human vein tissue for ultra performance liquid chromatography-mass spectrometry and nuclear magnetic resonance-based untargeted metabolic profiling

Human vein tissue is an important matrix to examine when investigating vascular diseases with respect to understanding underlying disease mechanisms. Here, we report the development of an extraction protocol for multi-platform metabolic profiling of human vein tissue. For the first stage of the optimization, two different ratios of methanol/water and 5 organic solvents--namely dichloromethane, chloroform, isopropanol, hexane and methyl tert-butyl ether (MTBE) solutions with methanol--were tested for polar and organic compound extraction, respectively. The extraction output was assessed using (1)H Nuclear Magnetic Resonance (NMR) spectroscopy and a panel of Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) methodologies. On the basis of the reproducibility of extraction replicates and metabolic coverage, the optimal aqueous (methanol/water) and organic (MTBE/methanol) solvents identified from the first stage were used in a sequential approach for metabolite extraction, altering the order of solvent-mixture addition. The combination of organic metabolite extraction with MTBE/methanol (3 : 1) followed by extraction of polar compounds with methanol/water (1 : 1) was shown to be the best method for extracting metabolites from human vein tissue in terms of reproducibility and number of signals detected and could be used as a single extraction procedure to serve both NMR and UPLC-MS analyses. Molecular classes such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines, sphingolipids, purines, and pyrimidines were reproducibly extracted. This study enabled an optimal extraction protocol for robust and more comprehensive metabolome coverage for human vein tissue. Many of the physiological and pathological processes affecting the composition of human vein tissue are common to other tissues and hence the extraction method developed in this study can be generically applied.

A comprehensive characterisation of the metabolic profile of varicose veins; implications in elaborating plausible cellular pathways for disease pathogenesis

Metabolic phenotypes reflect both the genetic and environmental factors which contribute to the development of varicose veins (VV). This study utilises analytical techniques to provide a comprehensive metabolic picture of VV disease, with the aim of identifying putative cellular pathways of disease pathogenesis. VV (n = 80) and non-VV (n = 35) aqueous and lipid metabolite extracts were analysed using 600 MHz 1H Nuclear Magnetic Resonance spectroscopy and Ultra-Performance Liquid Chromatography Mass Spectrometry. A subset of tissue samples (8 subjects and 8 controls) were analysed for microRNA expression and the data analysed with mirBase (www.mirbase.org). Using Multivariate statistical analysis, Ingenuity pathway analysis software, DIANALAB database and published literature, the association of significant metabolites with relevant cellular pathways were understood. Higher concentrations of glutamate, taurine, myo-inositol, creatine and inosine were present in aqueous extracts and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in lipid extracts in the VV group compared with non-VV group. Out of 7 differentially expressed miRNAs, spearman correlation testing highlighted correlation of hsa-miR-642a-3p, hsa-miR-4459 and hsa-miR-135a-3p expression with inosine in the vein tissue, while miR-216a-5p, conversely, was correlated with phosphatidylcholine and phosphatidylethanolamine. Pathway analysis revealed an association of phosphatidylcholine and sphingomyelin with inflammation and myo-inositol with cellular proliferation.

Venous Reflux Duplex Ultrasound Objective Structured Assessment of Technical Skills (V-DUOSATS)

Background: Treatment of a symptomatic incompetent saphenous vein is routinely accomplished using radiofrequency ablation (RFA). Treatment efficacy is dependent upon, among other factors, effective exsanguination of the target vein. Trendelenburg positioning is often used to facilitate emptying the vein. However, the angle of torso inversion is limited to approximately 15 due to patient stability in a head down position. In November 2007, our clinic implemented a technique whereby the patient’s leg was elevated to a consistent 30 angle while maintaining the torso horizontal, thus achieving enhanced vein emptying. Methods: Patient records for treatments prior to (May-October 2007; Group 1) and subsequent to (November 2007-December 2012; Group 2) implementing the enhanced vein drainage technique were retrospectively analyzed for outcomes. The VNUS Medical Technologies (Covidien, San Jose, Calif) ClosureFast catheters were used for all treatments. Results: Table I summarizes the patient demographics. Incomplete vein obliteration at first postoperative ultrasound was observed in one of 105 (1.0%) treatments in Group 1 (G1), compared with two of 895 (0.2%) in Group 2 (G2; P 1⁄4 NS). Documentation of an early first postoperative ultrasound was lacking for one G2 patient. For outcome analysis, treatments for which an ultrasound follow-up examination was not performed beyond 1 month were excluded. Results are shown in Table II. As would be expected since the G2 patients were treated more recently than the G1 patients, the months of follow-up were skewed toward a shorter interval in G2. In November 2009, we altered our technique from performing two heating passes in the proximal vein segment to a single pass. In comparing G2 treatments that received proximal vein double heating (n 1⁄4 161) to the G1 technique having a similar proximal heating protocol, it was found that one of the six G2 recanalizations occurred in this sub-group (P 1⁄4 .0404). The difference in outcomes between the G2 sub-groups, 1/161 (0.6%) vs 5/ 356 (1.4%), was not significant. Conclusions: An analysis of our clinical experience, although retrospective in nature, suggests that using enhanced leg elevation during saphenous vein RFA, by facilitating exsanguination, may be an effective technique to achieve durable vein obliteration.

Differential Metabolic Phenotype of Human Varicose Veins Tissue and Their Utility in Understanding Disease Pathogenesis and Identifying Potential Prognostic Biomarkers

anesthesia. Two studies were conducted to evaluate the feasibility of a novel biodegradable implant, polyglycolic acid (PGA), to occlude incompetent great saphenous veins (GSV) without tumescent anesthesia. Methods: TAHOE I and TAHOE II were prospective, single-arm studies using the same PGA implant. Slight modifications were made to the TAHOE II protocol (eg, removed mandate for heparin use and postprocedure compression). TAHOE I was conducted at three sites in Europe and enrolled 51 patients; TAHOE II was conducted at one site in the Dominican Republic and enrolled 30 patients. After treatment patients returned at 1 day, 1, 2, and 6 weeks, and 3 and 6 months. Vein occlusion, reflux, postprocedure pain (0-10), quality-of-life (CIVIQ2), Venous Clinical Severity Score (VCSS), and adverse events were assessed at each visit. Results: Occlusion and reflux-free rates are summarized in Table I. Initial occlusion and reflux-free rates were >90% in both studies. Change in CIVIQ2 scores and VCSS scores to 6 months are summarized in Table II. CIVIQ2 scores were elevated at 1 day but showed improvement at 6 weeks that was sustained through 6 months. VCSS improved after day 1 and through to 6 months. Pain (median [IQR]) at day 1 was 2.0 (0,3) in the TAHOE I study and 2.5 (0,5) in the TAHOE II study but decreased to 0 (0,0) from 2 weeks to 6 months for both groups. The most commonly reported adverse events were induration (20% TAHOE I, 41.4% TAHOE II), erythema (12% TAHOE I, 48.3% TAHOE II), fever (34.5% TAHOE II), nausea (34.5% TAHOE II), and phlebitis (8% TAHOE I); all resolved by 6 weeks. No patient experienced neuropraxia. Conclusions: Tumescent-free PGA implantation resulted in high initial GSV occlusion with recanalization appearing in some patients at 3 months postprocedure. PGA is promising, but requires modification to achieve higher long-term occlusion and reflux-free rates.